Project Summary: The long-term goal of this project is to investigate the mechanisms that activate the regenerative potential in adult cells. These processes occur naturally in animals that regenerate as adults, such as the Mexican Axolotl. By understanding how mature axolotl cells participate in a regenerative response, we will be able to determine whether similar mechanisms occur under natural or induced conditions in human cells. To identify the potential mechanisms underlying the changes that occur in Axolotl limb cells that make them capable of participating in regeneration, we will take a multipronged approach. In AIM 1 we will identify which factors in the microenvironment surrounding the limb cells makes them plastic, or able to change their cell identity. We will focus on nerve signaling and the signaling molecules FGF2, FGF8 and BMP2 that when applied together to the cells can substitute for the lack of nerves in regenerating tissue. We will also focus on the wound healing response, which is a requirement of regeneration. We have designed a new regeneration assay that will test whether these factors are sufficient to induce a regenerative response when coupled with another treatment (retinoic acid treatment). We will also characterize the expression of genes that play an important role in regeneration to see whether their expression is increased in the treated tissues. In AIM2 we will focus on how the factors identified in AIM1 make the limb cells capable of regenerating. We are focusing on chromatin modifications, which ?open up? the nuclear landscape and allow more genes, potentially those involved with regeneration, to be activated. In our pilot studies we have found that a large-scale opening up of the chromatin occurs. We have additionally observed that the abundance of an inhibitory chromatin mark, H3K27me3 (trimethylation of Lysine residue 27 of histone 3), decreases globally and on specific limb regeneration genes in cells as they participate in regeneration. Thus, we will test whether the factors in AIM1 alter the amount of H3K27 on limb regeneration genes (HoxA9, HoxD10, and HoxD11) specifically in connective tissue cells, which are the cells responsible for generating the missing blueprint, or pattern, of the regenerated structure. We will also test whether inhibition of the PRC2 pathway, which is responsible for H3K27me3, is required to make limb cells capable of regenerating. The overall strength of our proposal is the utilization of classical embryological manipulations in combination with new technologies, paired with phenomenological, molecular, and epigenetic assays to probe the molecular mechanisms underlying the natural regenerative response.